Discovery and Characterization of Nonpeptidyl Agonists of the Tissue-Protective Erythropoietin Receptor

Abstract

Erythropoietin (EPO) and its receptor are expressed in a wide variety of tissues, including the central nervous system. Local expression of both EPO and its receptor is upregulated upon injury or stress and plays a role in tissue homeostasis and cytoprotection. High-dose systemic administration or local injection of recombinant human EPO has demonstrated encouraging results in several models of tissue protection and organ injury, while poor tissue availability of the protein limits its efficacy. Here, we describe the discovery and characterization of the nonpeptidyl compound STS-E412 (2-[2-(4-chlorophenoxy)ethoxy]-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidine), which selectively activates the tissue-protective EPO receptor, comprising an EPO receptor subunit (EPOR) and the common β-chain (CD131). STS-E412 triggered EPO receptor phosphorylation in human neuronal cells. STS-E412 also increased phosphorylation of EPOR, CD131, and the EPO-associated signaling molecules JAK2 and AKT in HEK293 transfectants expressing EPOR and CD131. At low nanomolar concentrations, STS-E412 provided EPO-like cytoprotective effects in primary neuronal cells and renal proximal tubular epithelial cells. The receptor selectivity of STS-E412 was confirmed by a lack of phosphorylation of the EPOR/EPOR homodimer, lack of activity in off-target selectivity screening, and lack of functional effects in erythroleukemia cell line TF-1 and CD34(+) progenitor cells. Permeability through artificial membranes and Caco-2 cell monolayers in vitro and penetrance across the blood-brain barrier in vivo suggest potential for central nervous system availability of the compound. To our knowledge, STS-E412 is the first nonpeptidyl, selective activator of the tissue-protective EPOR/CD131 receptor. Further evaluation of the potential of STS-E412 in central nervous system diseases and organ protection is warranted.

Fig. 1.

RhEPO and STS-E412 increase EPOR phosphorylation in primary cultures of human cortical cells. (A) Increase in EPOR phosphorylation in response to increasing concentrations of rhEPO. (B) Increase in EPOR phosphorylation in response to increasing concentration of STS-E412. Unfilled bars represent basal phosphorylation in the absence of rhEPO or STS-E412. Solid bars (A) represent pEPOR levels measured 12 minutes after the addition of rhEPO at the indicated concentrations (IU/ml), and gray bars (B) represent phosphorylation measured 12 minutes after the addition of the indicated concentrations of STS-E412 (in nanomolar). The pEPOR levels were determined by an ELISA as described in Materials and Methods. The results from three independent experiments (means ± S.E.M.) using three different human cortical culture preparations are presented normalized to the respective control values. Each experiment was performed using one to three biologic replicates. Basal pEPOR levels were 0.33 ± 0.13 pg/μg (mean ± S.D., range: 0.23–0.47 pg/μg protein). *P < 0.05 compared with the control (basal phosphorylation) according to an unpaired Student’s t test.

Fig. 2.

STS-E412 activates the EPOR/CD131 receptor but not the EPOR/EPOR homodimer. Phosphorylation of the EPOR, CD131, and the signaling molecules JAK2 and AKT were studied in HEK293 cells coexpressing the EPOR and CD131 (A–D) or in HEK293 cells expressing only the EPOR (E). In all the figures, the unfilled bars represent basal phosphorylation in the absence of rhEPO or STS-E412, solid bars represent phosphorylation levels measured 12 minutes after the addition of rhEPO (10 IU/ml), and gray bars represent phosphorylation measured 12 minutes after the addition of the indicated concentrations of STS-E412 (in nanomolar). Phosphoprotein levels were determined by an ELISA as described in Materials and Methods. (A) Increase in pEPOR induced by rhEPO and STS-E412. The data represent increases in pEPOR (mean ± S.E.M.) from five independent experiments; each experiment included three to six replicate measurements. Basal pEPOR levels were 0.66 ± 0.25 pg/μg (mean ± S.D.). (B) Increase in pCD131 induced by rhEPO and STS-E412. The data are representative of three independent experiments (mean ± S.E.M.; each experiment included two to three biologic replicates). (C) Increase in pY¹⁰⁰⁷pY¹⁰⁰⁸-JAK2 induced by rhEPO and STS-E412. The data are representative of two independent experiments, each performed in two or eight biologic replicates. (D) Increase in pS⁴⁷³-AKT induced by rhEPO and STS-E412. The data are representative of three independent experiments each performed in two to eight biologic replicates (mean ± S.E.M.). (E) STS-E412, in contrast to rhEPO, does not increase EPOR phosphorylation in HEK293 cells or HEK293 cells overexpressing EPOR alone. The data represent averaged increases in pEPOR from triplicate measurements under control conditions (unfilled bars), in the presence of rhEPO (solid bars), or in the presence of STS-E412 (gray bars). The cells were exposed to rhEPO or STS-E412 at the indicated concentrations for 12 minutes, and the reaction was quenched by the addition of a lysis buffer. Similar results were obtained in five independent experiments and at concentrations of STS-E412 ranging from 1 to 60,000 nM. *P < 0.05 compared with the control (Ctrl) according to Student’s t test.

Fig. 3.

Effects of rhEPO and STS-E412 on proliferation of TF-1 and primary human CD34⁺ cells. TF-1 cells (A and B) were starved of IL-3 for 24 hours before the experiment. Then rhEPO (A) or STS-E412 (B) were added to the cells at the indicated concentrations, and the cells were returned to a humidified 37°C CO₂ incubator for 72 hours. Proliferation was measured by increases in ATP using a luminescence detection assay (CellTiter-Glo; Promega). The data represent the results of six (STS-E412) or eight (control, rhEPO) replicate cultures (mean ± S.E.M.). The results are representative of three independent experiments. (C and D) Effects of (C) rhEPO and (D) STS-E412 on the proliferation of primary human CD34⁺ cells. We added rhEPO or STS-E412 to yield the indicated concentrations, and the cells were returned to a humidified 37°C incubator with 5% CO₂ for 10 days. Proliferation was measured by increases in ATP using a luminescence detection assay (CellTiter-Glo; Promega). The data represent the mean ± S.E.M. of two independent experiments, each performed in triplicate and normalized to the respective control values. Controls are represented by unfilled bars; rhEPO is represented by solid bars, and STS-E412 is represented by gray bars. RLU, relative luminescence units. *P < 0.05 compared with the control according to Student’s t test.

Fig. 4.

STS-E412 protects primary human and rat neuronal cells from cytotoxic insults. (A) Primary human cortical cells were pretreated with STS-E412 at the indicated concentrations or rhEPO (10 IU/ml) for 24 hours before a staurosporine insult (500 nM). After culturing the cells for an additional 24 hours, the supernatants were harvested, and the LDH levels were measured as described in the Materials and Methods section. The data represent the mean ± S.E.M. of six (rhEPO, STS-E412) or nine (control [Ctrl], staurosporine alone) replicates. (B) Primary rat hippocampal cells were pretreated with STS-E412 at the indicated concentrations or rhEPO (10 IU/ml) for 24 hours before a glutamate insult (100 μM). The cells were then cultured for an additional 24 hours. The supernatants were harvested, and the LDH levels were measured as above. *P < 0.05 compared with the control according to Student’s t test.

Fig. 5.

STS-E412 protects primary human RPTEC from H₂O₂-mediated cytotoxicity. RPTEC were pretreated with STS-E412 or rhEPO at the indicated concentrations for 24 hours before H₂O₂ insult (150 μM). The cells were then treated with H₂O₂ and returned to a 37°C 5% CO₂ incubator for another 24 hours. CellTiter Glo (A) and LDH assays (B) were then performed. Unfilled bars indicate control values (insult only); solid bars represent measured values in the presence of rhEPO (10 IU/ml), and gray bars indicate measured values in the presence of STS-E412 at the indicated concentrations. LDH and ATP levels were measured as described in Materials and Methods. The data represent the mean ± S.E.M. of six (control [Ctrl], H₂O₂ alone) or three (rhEPO, STS-E412) replicates. *P < 0.05 compared with the control according to Student’s t test.